Enzyme-sensitive therapeutic wound dressings

ABSTRACT

The invention provides a wound dressing comprising a therapeutic agent and a matrix comprising polymers joined by cross-linkages which cross-linkages comprise oligopeptidic sequences which are cleavable by a protease associated with wound fluid such that the rate of release of the therapeutic agent increases in the presence of the protease.

The present invention relates to wound dressing materials, and inparticular to new materials for the controlled release of therapeuticagents into wounds.

All publications, patents and patent applications cited herein areincorporated in full by reference.

In mammals, injury triggers an organised complex cascade of cellular andbiochemical events that result in a healed wound. Wound healing is acomplex dynamic process that results in the restoration of anatomiccontinuity and function; an ideally healed wound is one that hasreturned to normal anatomic structure, function and appearance.

Infection of wounds by bacteria delays the healing process, sincebacteria compete for nutrients and oxygen with macrophages andfibroblasts, whose activities are essential for the healing of thewound. Infection results when bacteria achieve dominance over thesystemic and local factors of host resistance. Infection is therefore amanifestation of disturbed host/bacteria equilibrium in favour of theinvading bacteria. This elicits a systemic septic response, and alsoinhibits the multiple processes involved in wound healing. Lastly,infection can result in a prolonged inflammatory phase and thus slowhealing, or may cause further necrosis of the wound. The granulationphase of the healing process will begin only after the infection hassubsided.

Chronically contaminated wounds all contain tissue bacterial flora.These bacteria may be indigenous to the patient or might be exogenous tothe wound. Closure or eventual healing of the wound is often based on aphysician's ability to control the level of the bacterial flora.

If clinicians could respond to wound infection as early as possible theinfection could be treated topically as opposed to having to useantibiotics. This would also lead to less clinicalintervention/hospitalisation and would reduce the use of antibiotics andother complications of infection.

Current methods used to identify bacterial infection rely mainly onjudgement of the odour and appearance of a wound. With experience, it ispossible to identify an infection in a wound by certain chemical signssuch as redness or pain. Some clinicians take swabs that are thencultured in the laboratory to identify specific organisms, but thistechnique takes time.

Pain is also associated with infected and chronic wounds. Biochemically,pain is experienced when there is an increase of kinins (bradykinin) inthe area of the wound. Kinins are produced by the proteolytic breakdownof kininogen, and the protease responsible for this is kallikrein.Kallikrein also stimulates the production of tissue plasminogenactivator (t-PA).

It has now been discovered that wound fluid from wounds that areapparently not clinically infected but which go on to become infectedwithin a few days have elevated levels of neutrophil elastase activityand may also have high levels of other inflammatory enzymes, such asmacrophage proteases, other neutrophil proteases, bacterial collagenase,plasmin, hyaluronidase, kallikrein or t-PA.

Further, chronic wounds, such as venous ulcers, pressure sores anddiabetic ulcers have a disordered wound-healing metabolism even in theabsence of infection. In particular, wound chronicity is associated withelevated levels of protease enzymes in the wound that interfere with thenormal processes of tissue formation and destruction in the wound.

It is known to provide antimicrobial wound dressings. For example, suchdressings are known having a liquid permeable wound contacting layer, anintermediate absorbent layer and an outer, liquid-impervious backinglayer, in which one or more of the layers contains an antimicrobialagent. For example, EP-A-0599589 describes layered wound dressingshaving a wound contacting layer of a macromolecular hydrocolloid, anabsorbent layer, and a continuous, microporous sheet intermediate thewound contacting layer and the absorbent layer. The absorbent layercontains a low molecular weight antimicrobial agent that can diffuseinto the wound.

WO-A-0238097 describes wound dressings comprising a liquid-permeable topsheet having a wound facing surface and a back surface, and a hydrogellayer on the wound-facing surface of the top sheet. The top sheet isadapted to block or restrict passage of liquid from the back surface tothe wound-facing surface. The hydrogel layer is an insoluble hydrogeladapted to maintain a moist wound-healing environment at the woundsurface. The hydrogel may contain therapeutic agents, such asantimicrobial agents, for sustained release into the wound.

Previous antimicrobial wound dressings suffer from the drawback that therelease of the antimicrobial agent is relatively unresponsive to thedegree of infection of the wound being treated. This is undesirablebecause it can result in resistant microorganisms, and also because allunnecessary medication can interfere with the processes of woundhealing.

A first aspect of the invention provides a wound dressing comprising atherapeutic agent and a matrix comprising polymers joined bycross-linkages which cross-linkages comprise, or consist of,oligopeptidic sequences which are cleavable by a protease associatedwith wound fluid such that the rate of release of the therapeutic agentincreases in the presence of the protease.

Preferably, the matrix consists of the cross-linked polymers andoptionally also the therapeutic agent.

Preferably, the protease is associated with a wound healing disorder,e.g. Infection, or ulcer formation (wound chronicity). In this way, therate of release of the therapeutic agent may increase if the wound isinfected or is a chronic wound.

By an “increase” in the rate of release of the therapeutic agent weinclude the situation where the rate of release of the therapeutic agentincreases by at least 1.5, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10- or15-fold. Preferably, there is no release of the therapeutic agent in theabsence of the protease.

By “a protease associated with wound infection” we include proteasesthat are elevated during infection and proteases that are elevated inwounds that are apparently not clinically infected but which go on tobecome infected within a few days. Similarly, by “a protease associatedwith ulcer formation” we include proteases that are elevated in chronicwounds.

The principle underlying the present invention is that the cross-linkedpolymers would behave as both an enzyme sensor and as anenzyme-dependent delivery system. In the absence of the target proteasethe oligopeptidic sequences remain intact, keeping the pore size smalland preventing (or at least keeping to low levels) the release of thetherapeutic agent. Elevated protease levels (e.g. in wound infection orwound chronicity) hydrolyse the oligopeptidic sequences which results inincreased pore size and permeability. The therapeutic agent is thenreleased from the dressing so that it is free to migrate into the wound.In this way, delivery of the therapeutic agent increases in the presenceof the protease so that if the wound is infected (including as indicatedabove when a protease associated with infection is elevated in a woundthat is apparently not clinically infected but which goes on to becomeinfected within a few days) or is a chronic wound delivery of thetherapeutic agent increases.

The term “polymer” as used herein includes homopolymers and copolymers(e.g. random copolymers, alternating copolymers and block copolymers).

Although polymers which are degraded by the target protease could beused, it is preferred that the polymers are not degraded by the targetprotease or other factors (e.g. other proteases) that may be present inthe wound environment.

In theory, any polymer containing groups to which the reactive groupscan be attached may be used, although of course the skilled person willappreciate that considerations such as toxicity should be taken intoaccount. Similarly, the polymers used should not be immunogenic.

In selecting a polymer, charge and size may also be important as anincrease in crystallinity will increase order and therefore reducepermeability of barrier. The longer the polymers the more likely theyare to become physically intertwined, and consequently the less likelythey are to fall apart. In view of this it is preferred that shortpolymers (i.e. 5 to 50 monomers) are used.

Preferably, a polyfunctional polymer is used as the pore size will besmaller and the ability to retain the therapeutic agent in the absenceof protease will be higher.

Preferably, the polymers are non-ionic surfactants, polyalkoylatedalcohols, alkyl or dialkyl polyglycerol compounds, polyethyloxylatedalcohols, polymers (including homopolymers and copolymers) of acrylamide(e.g. N-(2-hydroxypropyl)methacrylamide (HPMA)), polynucleotides,polypeptides or carbohydrates.

Preferably, the polymers are synthetic polymers. Examples of syntheticpolymers include polyvinyl alcohol, polyethylene glycerol, PVP,polyolefins, fluoropolymers, hydropolymers from vinyl esters, vinylethers, carboxy vinyl monomers, meth(acrylic) acid, acrylamide, N-vinylpyrrolidone, acylamidopropanem acylamidopropane, PLURONIC (Maleic acid,NN-dimethylacrylamide diacetone acrylamide acryloyl, morpholine andmixtures thereof, and oxidized regenerated cellulose.

Alternatively, natural polymers such as carbohydrates (e.g. dextan,chitin or chitosan) natural peptides or proteins (collagens, elastin,fibronectins, or even soluble proteins such as albumin), or semisynthetic peptides (made by using a peptide synthesizer or byrecombinant techniques) may be used.

In a preferred embodiment, polymers ofN-(2-hydroxypropyl)methyacrylamide (HPMA) are used. In this regard,reference is made to Ulbrich et al. (1980) Biomaterials 1, 199-204,which details the crosslinking of HPMA polymers by peptides.

As mentioned above, the polymers are joined by cross-linkages whichcomprise cleavable oligopeptidic sequences. Oligopeptides are generallydefined as polypeptides of short length, typically twenty amino acids orfewer. Preferably, the oligopeptidic sequences employed in the presentinvention consist of 3 to 15 amino acids, preferably 3 to 10 aminoacids, more preferably, 3 to 8 amino acids and yet more preferably 4 to8 amino acids. Preferably, the oligopeptidic sequences consist of 3, 4,5, 6, 7 or 8 amino acids.

The degree of crosslinking of the polymers should be sufficient suchthat the rate of release of the therapeutic agent increases in thepresence of the protease. Preferably, the degree of crosslinking of thepolymers should be sufficient to render the matrix sufficientlyimpermeable to the molecule to be delivered so that the therapeuticagent is only released in the presence of the target protease. This willbe dependent on the molecular weight of the therapeutic agent.

The rate of degradation of the matrix will depend on a number offactors, including the length of the oligopeptidic sequences. Ulbrich etal. noted that extension of the peptidic linkers by one amino acidresidue to give a peptidic linker of four amino acids caused apronounced rise in the rate of cleavage of the polymeric substrates.Ulbrich et al. reported that extension of the oligopeptidic sequence ledto a decrease in the steric hindrance by polymer chain and thus to anincrease in degradability.

Steric hindrance may also be reduced by coupling the oligopeptidicsequence to the polymer by means of an appropriate spacer. Thus, theoligopeptidic sequences may couple the polymers directly (in which casethe cross-linkage consists of the oligopeptidic sequence) or by means ofan appropriate spacer.

The following paper gives a useful review of bioconjugation techniquesfor use in pharmaceutical chemistry: Veronese, F. M. and Morpurgo, M(1999) Bioconjugation in Pharmaceutical chemistry II Farmaco, 54,497-516. This paper describes in detail the chemistry of each amino acidand which ones are most suitable for use in bioconjugation techniques.For example, it demonstrates that conjugation would occur by nucleophileto electrophile attacks. The amino acid side chains R—S—, R—NH2, R—COO—and ═R—O— are well suited to bioconjugation (to natural or syntheticmolecules).

In addition this paper indicates and gives examples of a wide range ofstructures and chemical groups that the peptides (containing amino (e.g.lysine), carboxyl (COO—) or cystyl groups (R—SH) can bind to.

With regard to conjugation techniques, see also Ulbrich, K., et al(2000) Polymeric drugs based on conjugates of synthetic and naturalmarcomolecules I. Synthesis and physico-chemical characterisation.Journal of controlled release 64, 63-79. This reference describes howantibodies, peptides or proteins can be conjugated to synthetic polymers(e.g. poly HPMA).

The rate of degradation will not only depend on the number of aminoacids but also on the nature of the amino acids comprising thecross-links. This dependency arises from the substrate specific natureof proteases. The region of the enzyme where interaction with thesubstrate takes place is known as the “active site” of the enzyme. Theactive site performs the dual role of binding the substrate whilecatalysing the reaction, for example cleavage. Studies of the structuresof the complexes of proteolytic enzymes with peptides indicate that theactive site of these enzymes is relatively large and binds to severalamino acid residues in the peptide. Thus, the degradability of aparticular bond in a peptide chain depends not only on the nature of thestructure near the cleaved bond, but also on the nature of the aminoacid residues which are relatively remote from the cleaved bond, butplay an important part in holding the enzyme in position duringhydrolysis.

The structure of the oligopeptidic sequences must be chosen so as tocorrespond to that of the active site of the protease responsible forthe degradation. The protease may be a host-derived protease or aprotease produced by pathogens (e.g. bacteria) at the site of infection.Examples of such enzymes include, but are not restricted to: matrixmetalloproteinases and other extracellular matrix component proteases(including collagenases, stromelysins, matrilysin, gelatinases andelastases), lysosomal enzymes (including cathepsin), serine proteasesand other enzymes of the clotting cascade (such as thrombin), enzymes ofthe endoplasmic reticulum (such as cytochrome P450 enzymes, hydrolyticreaction enzymes and conjugation reaction enzymes), non-specificaminopeptidases and esterases, carboxypeptidases, phosphatases, andglycolytic enzymes. Thrombin-like, alanine aminopeptidase, andelastase-like enzymatic activity are common in bacterial infections, andthe amino acid cleavage sequences of such enzymes are well-documented.WO 00/64486 discloses amino acid cleavage sequences of various enzymesassociated with wound infection.

Preferably, the protease is a macrophage or neutrophil protease, or ahuman or bacterial collagenase or gelatinase. The macrophage andneutrophil proteases include elastase, matrix metalloproteinase 9(MMP-9), MMP-8, cathepsin G, MMP-12, capases and mixtures thereof.

Preferably, the protease is a collagenase, gelatinase, elastase, matrixmetalloproteinase, stromelysin, cathepsin G, thrombin or capase.

In one embodiment, the protease is elastase and the oligopeptidicsequence comprises or consists of lys-gly-ala-ala-ala-lys,-Ala-Ala-Ala-, Ala-Ala-Pro-Val, Ala-Ala-Pro-Leu, Ala-Ala-Pro-Phe,Ala-Ala-Pro-Ala or Ala-Tyr-Leu-Val.

Preferably, the oligopeptidic sequence is cleavable by elastase but isnot cleavable by a MMP such as MMP-2 or MMP-9.

In another embodiment, the protease is a matrix metalloproteinase andthe oligopeptidic sequence comprises or consists of-Gly-Pro-Y-Gly-Pro-Z-, -Gly-Pro-Leu-Gly-Pro-Z-, -Gly-Pro-Ile-Gly-Pro-Z-,or -Ala-Pro-Gly-Leu-Z-, where Y and Z are amino acids where Y and Z areamino acids.

In another embodiment, the protease is a collagenase and theoligopeptidic sequence comprises or consists of-Pro-Leu-Gly-Pro-D-Arg-Z-, -ProLeu-Gly-Leu-Leu-Gly-Z-,-Pro-Gln-Gly-Ile-Ala-Gly-Trp-, -Pro-Leu-Gly-Cys(Me)-His-,-Pro-Leu-Gly-Leu-Trp-Ala-, -Pro-Leu-Ala-Leu-Trp-Ala-Arg-, or-Pro-Leu-Ala-Tyr-Trp-Ala-Arg-, where Z is an amino acid.

In another embodiment, the protease is a gelatinase and theoligopeptidic sequence comprises or consists of-Pro-LeuGly-Met-Trp-Ser-Arg-.

In another embodiment, the protease is a thrombin and the oligopeptidicsequence comprises or consists of -Gly-Arg-Gly-Asp-, -Gly-Gly-Arg-,-Gly-Arg-Gly-Asp-Asn-Pro-, -Gly-Arg-Gly-Asp-Ser-,-Gly-Arg-Gly-Asp-Ser-Pro-Lys-, -Gly-Pro-Arg-, -Val-Pro-Arg-, or-Phe-Val-Arg-.

In another embodiment, the protease is a stromelysin and theoligopeptidic sequence comprises or consists of-Pro-TyrAla-Tyr-Trp-Met-Arg-.

In one preferred embodiment of the invention, the protease is elastaseand the polymers are HPMA polymers and the oligopeptidic sequencescomprise or consist of lys-gly-ala-ala-ala-lys, -Ala-Ala-Ala-,Ala-Ala-Pro-Val, Ala-Ala-Pro-Leu, Ala-Ala-Pro-Phe, Ala-Ala-Pro-Ala orAla-Tyr-Leu-Val. Preferably, the oligopeptidic sequences directly couplethe HPMA polymers (i.e. without the presence of spacers). In thisexample the terminal lysines may be added to bind the peptide to thepolymer.

Preferably, the oligopeptidic sequences are cleavable by only oneprotease associated with wound fluid, preferably elastase.Alternatively, the oligopeptidic sequences may be cleavable by two,three or more proteases associated with wound fluid.

The design of the linking oligopeptidic sequence is important as it mustnot only contain a hydrolysable sequence that would be cleaved in thepresence of the protease but also a terminal amino acid that can bereadily conjugated to the polymers employed or to a spacer. Examples ofreactive amino acids that could be used to link the oligopeptidicsequences to the polymers or spacers include cysteine and lysine.

The therapeutic agent may, for example, be an antimicrobial agent and/ora pain relieving agent. The antimicrobial agent may, for example,comprise an antiseptic, an antibiotic, or mixtures thereof.

In selecting one or more therapeutic agents for use with the wounddressings of the present invention, it is preferred that largermolecules are employed (e.g. molecules having a molecular weight of atleast 500, 1,000, 5,000, 10,000, or 20,000). Small molecules maypenetrate the matrix, whereas larger molecules such as chlorohexidinemay be better suited to this type of application. Bioerodible particlesand colloidal silver are suitable. Although it is preferred that thetherapeutic agent is one which cannot penetrate the matrix, it shouldnevertheless be appreciated that small molecules such as silver saltsmay be employed since the release of such therapeutic agents may stillbe to some extent responsive to levels of protease as in the presence ofelevated levels of the target protease the cross-linkages which becleaved which will result in an increase in the rate of release of thetherapeutic agent. Further, if a polyfunctional polymer is used the poresize of the matrix will be smaller and thus the ability of the matrix toretain the therapeutic agent in the absence of protease will be higher.Moreover, as noted above, the degree of cross-linking will influence thepermeability of the matrix.

Preferred antibiotics include peptide antimicrobials (e.g. defensins,Magainin, synthetic derivatives of them) tetracycline, penicillins,terramycins, erythromycin, bacitracin, neomycin, polymycin B, mupirocin,clindamycin and mixtures thereof. Preferred antiseptics include silversulfadiazine, chlorhexidine, povidone iodine, triclosan, other silversalts, sucralfate, quaternary ammonium salts and mixtures hereof. Thepain relieving agent may be an analgesic or a local anaesthetic.

The therapeutic agent may be incorporated within the matrix of theinvention or may alternatively be located behind the matrix in a “donorlayer”. Thus, in one embodiment of the first aspect invention, thetherapeutic agent is incorporated within the matrix. For ready releaseof the therapeutic agent upon elevation of the target protease, thetherapeutic agent should not covalently bound to the matrix. Forexample, if the molecule to be delivered is relatively inert it could bemixed into the formulation during manufacture. Silver is one example ofa molecule that could be delivered in this way. The wound contactinglayer of the dressing may comprise or consist of the matrix in which thetherapeutic agent has been incorporated into. Alternatively, thedressing may comprise a liquid permeable wound contacting layer, anintermediate layer (which may be an absorbent layer) comprising orconsisting of the matrix within which the therapeutic agent has beenincorporated and an outer, liquid-impervious backing layer. Upondegradation of the matrix by proteases present in wound fluid, thetherapeutic agent present in the intermediate layer may diffuse into thewound.

Another embodiment of the first aspect of the invention provides a wounddressing which comprises a barrier layer which comprises thecross-linked matrix of the invention, the barrier layer being forinitially separating the therapeutic agent in the wound dressing fromwound fluid when in use. Suitably, the barrier layer consists of thematrix.

The barrier layer is separate from the therapeutic agent, and thetherapeutic agent is initially prevented from contacting the wound fluidby the barrier layer. That is to say, the bioavailability of thetherapeutic agent to the wound surface is low until the peptidecross-linkages in the barrier material have been broken down by theenzyme, at which point the bioavailability of the therapeutic agentincreases. Since protease levels are elevated in chronic and infectedwounds, this provides for accelerated and/or selective release of thetherapeutic agent into such wounds. The barrier layer is normallysubstantially impervious to wound fluid and insoluble therein unless thewound fluid contains a sufficient level of the specified enzyme to breakdown the substrate material.

The barrier layer is preferably about 0.1 to about 3 mm thick.Preferably about 0.5 to 1.5 mm thick. The cross-linked polymers may becombined in a film-forming composition with polymeric materials,plasticisers, and humectants. Suitable polymers include alginates, guargum, carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose, locust bean gum, carrageenan, chitosan, heparan sulfate,dermatan sulfate, glycosaminoglycans such as hyaluronic acid,proteoglycans, and mixtures thereof. Suitable plasticisers include C2-C8polyhydric alcohols such as glycerol. Preferably the cross-linkedpolymers make up at least about 10% by weight, more preferably at leastabout 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% by weight of thefilm-forming composition.

In certain embodiments the barrier layer comprises a substantiallycontinuous film comprising the film forming composition of thecross-linked polymers as described above.

In other embodiments the barrier layer comprises an apertured sheethaving a composition comprising the cross-linked polymers appliedthereto in occlusive fashion. The occlusive composition may be similarto the film-forming composition described above. In these embodiments,the apertures typically make up from about 0.1% to about 50% of the areaof the wound facing surface of the sheet before swelling, more typicallyfrom about 1% to about 30% of the area of the apertured sheet, andpreferably from about 10% to about 25% of the area of the aperturedsheet. Typically, the apertured sheet has from about 1 to about 30apertures per square cm, for example from about 4 to about 15 aperturesper square cm or from about 5 to about 10 apertures per square cm. Incertain embodiments the apertures are uniformly distributed over thesurface of the sheet, preferably in a regular pattern. The mean area ofeach aperture may for example be from about 0.01 to about 10 mm²,preferably from about 0.1 to about 4 mm², and more preferably from about1 mm² to about 2 mm². It will be appreciated that the sheet may includemore than one size and shape of aperture in order to provide aperturesthat open more or less quickly on exposure to infected wound fluid. Thisenables still more control over the dynamics of therapeutic agentdelivery to the wound. Typically, substantially the whole area of theapertures in the apertured sheet is blocked by the barrier materialbefore exposure to wound exudate.

Preferably, the thickness of the barrier film or the apertured sheet (byASTM D374-79) is from about 0.2 to about 5 mm, more preferably fromabout 0.4 to about 3 mm.

In one embodiment the barrier layer material may comprise, in additionto the cross-linked matrix of the invention, a polymer selected from thegroup consisting of water soluble macromolecular materials (hydrogels)such as sodium alginate, sodium hyaluronate, alginate derivatives suchas the propylene glycol alginate described in EP-A-0613692, and solublehydropolymers formed from vinyl alcohols, vinyl esters, vinyl ethers andcarboxy vinyl monomers, meth(acrylic) acid, acrylamide, N-vinylpyrrolidone, acylamidopropane sulphonic acid, PLURONIC (Registered TradeMark) (block polyethylene glycol, block polypropylene glycol)polystyrene-, maleic acid, NN-dimethylacrylamide diacetone acrylamide,acryloyl morpholine, and mixtures thereof. Suitable hydrogels are alsodescribed in U.S. Pat. No. 5,352,508.

In one embodiment the barrier layer material may comprise, in additionto the cross-linked matrix of the invention, a polymer selected from thegroup consisting of bioerodible polymers such aspolylactide/polyglycolide, collagen, gelatin, polyacrylate gels such asthose described in EP-A-0676457, calcium alginate gels, cross-linkedhyaluronate gels, gels of alginate derivatives such as propylene glycolalginate, and gels wherein the hydropolymer is formed from vinylalcohols, vinyl esters, vinyl ethers and carboxy vinyl monomers,meth(acrylic) acid, acrylamide, N-vinyl pyrrolidone, acylamidopropanesulphonic acid, PLURONIC (Registered Trade Mark) (block polyethyleneglycol, block polypropylene glycol) polystyrene-, maleic acid,NN-dimethylacrylamide diacetone acrylamide, acryloyl morpholine, andmixtures thereof. Suitable hydrogels are also described in U.S. Pat. No.5,352,508.

The barrier layer material may further comprise from about 5 to about50% by weight, preferably from 15 to 40% by weight, on the same basis ofone or more humectants such as glycerol. The barrier layer material mayfurther contain up to about 30% w/w, more preferably up to about 15% w/won the same basis of water.

The matrix of the invention comprising the therapeutic agent may contactthe barrier layer directly, or may be separated therefrom for example byan absorbent layer.

Preferably, the wound dressing of the invention comprises an absorbentlayer and/or a backing layer. As will be evident from the above, theabsorbent layer may, for example, separate the barrier layer from thetherapeutic agent containing cross-linked matrix or alternatively theabsorbent layer may comprise the therapeutic agent containingcross-linked matrix.

The area of the optional absorbent layer is typically in the range offrom 1 cm² to 200 cm², more preferably from 4 cm² to 100 cm².

The optional absorbent layer may comprise any of the materialsconventionally used for absorbing wound fluids, serum or blood in thewound healing art, including gauzes, nonwoven fabrics, superabsorbents,hydrogels and mixtures thereof. Preferably, the absorbent layercomprises a layer of absorbent foam, such as an open celled hydrophilicpolyurethane foam prepared in accordance with EP-A-0541391, the entirecontent of which is expressly incorporated herein by reference. In otherembodiments, the absorbent layer may be a nonwoven fibrous web, forexample a carded web of viscose staple fibers. The basis weight of theabsorbent layer may be in the range of 50-500 g/m², such as 100-400g/m². The uncompressed thickness of the absorbent layer may be in therange of from 0.5 mm to 10 mm, such as 1 mm to 4 mm. The free(uncompressed) liquid absorbency measured for physiological saline maybe in the range of 5 to 30 g/g at 25°.

Preferably, the wound dressing further comprises a backing layercovering the barrier sheet and the optional absorbent layer on the sideopposite the wound-facing side of the dressing. The backing layerpreferably provides a barrier to passage of microorganisms through thedressing and further preferably blocks the escape of wound fluid fromthe dressing. The backing layer may extend beyond at least one edge ofthe barrier sheet (if present) and optional absorbent layer to providean adhesive-coated margin adjacent to the said edge for adhering thedressing to a surface, such as to the skin of a patient adjacent to thewound being treated. An adhesive-coated margin may extend around allsides of the barrier sheet (if present) and optional absorbent layer, sothat the dressing is a so-called island dressing. However, it is notnecessary for there to be any adhesive-coated margin.

Preferably, the backing layer is substantially liquid-impermeable. Thebacking sheet is preferably semipermeable. That is to say, the backingsheet is preferably permeable to water vapour, but not permeable toliquid water or wound exudate. Preferably, the backing sheet is alsomicroorganism-impermeable. Suitable continuous conformable backingsheets will preferably have a moisture vapor transmission rate (MVTR) ofthe backing sheet alone of 300 to 5000 g/m²/24 hrs, preferably 500 to2000 g/m²/24 hrs at 37.5° C. at 100% to 10% relative humiditydifference. The backing sheet thickness is preferably in the range of 10to 1000 micrometers, more preferably 100 to 500 micrometers.

Suitable polymers for forming the backing sheet include polyurethanesand poly alkoxyalkyl acrylates and methacrylates such as those disclosedin GB-A-1280631. Preferably, the backing sheet comprises a continuouslayer of a high density blocked polyurethane foam that is predominantlyclosed-cell. A suitable backing sheet material is the polyurethane filmavailable under the Registered Trade Mark ESTANE 5714F.

The adhesive layer (where present) should be moisture vapor transmittingand/or patterned to allow passage of water vapor therethrough. Theadhesive layer is preferably a continuous moisture vapor transmitting,pressure-sensitive adhesive layer of the type conventionally used forisland-type wound dressings, for example, a pressure sensitive adhesivebased on acrylate ester copolymers, polyvinyl ethyl ether andpolyurethane as described for example in GB-A-1280631. The basis weightof the adhesive layer is preferably 20 to 250 g/m², and more preferably50 to 150 g/m². Polyurethane-based pressure sensitive adhesives arepreferred.

Preferably, the adhesive layer extends outwardly from the absorbentlayer and the envelope to form an adhesive-coated margin on the backingsheet around the absorbent layer as in a conventional island dressing.

Also within the scope of the present invention are embodiments in whichthe cross-linked matrix material substantially encapsulates thetherapeutic agent. For example, the dressing may comprise, or consistessentially of, particles such as microspheres of therapeutic agent(e.g. antimicrobial material) encapsulated in a layer comprising thecross-linked matrix material. The particles are preferably loaded withfrom 1 to 90 wt. %, more preferably from 3 to 50 wt. % of thetherapeutic agent.

The particles may be made by any suitable technique, includingcomminution, coacervation, or two-phase systems for example as describedin U.S. Pat. No. 3,886,084. Techniques for the preparation of medicatedmicrospheres for drug delivery are reviewed, for example, in PolymericNanoparticles and Microspheres, Guiot and Couvreur eds., CRC Press(1986).

A preferred method for preparation of the microparticles iscoacervation, which is especially suited to the formation of particlesin the preferred size range of 100 to 500 micrometers having a highloading of therapeutic agents. Coacervation is the term applied to theability of a number of aqueous-solutions of colloids, to separate intotwo liquid layers, one rich in colloid solute and the other poor incolloid solute. Factors which influence this liquid-liquid phaseseparation are: (a) the colloid concentration, (b) the solvent of thesystem, (c) the temperature, (d) the addition of anotherpolyelectrolyte, and (e) the addition of a simple electrolyte to thesolution. Coacervation can be of two general types. The first is called“simple” or “salt” coacervation where liquid phase separation occurs bythe addition of a simple electrolyte to a colloidal solution. The secondis termed “complex” coacervation where phase separation occurs by theaddition of a second colloidal species to a first colloidal solution,the particles of the two dispersed colloids being oppositely charged.Generally, materials capable of exhibiting an electric charge insolution (i.e. materials which possess an ionizable group) arecoacervable. Such materials include natural and synthetic macromolecularspecies such as gelatin, acacia, tragacanth, styrene-maleic anhydridecopolymers, methyl vinyl ether-maleic anhydride copolymers,polymethacrylic acid, and the like.

If, prior to the initiation of coacervation, a water-immisciblematerial, such as an oil, is dispersed as minute droplets in an aqueoussolution or sol or an encapsulating colloidal material, and then, asimple electrolyte, such as sodium sulfate, or another, oppositelycharged colloidal species is added to induce coacervation, theencapsulating colloidal material forms around each oil droplet, thusinvesting each of said droplets in a liquid coating of the coacervatedcolloid. The liquid coatings which surround the oil droplets mustthereafter be hardened by cross-linking to produce solid-walledmicrocapsules

Preferably, the wound dressing according to any aspect of the presentinvention is sterile and packaged in a microorganism-impermeablecontainer.

1. A wound dressing comprising a therapeutic agent and a matrixcomprising polymers joined by cross-linkages which cross-linkagescomprise, or consist of, oligopeptidic sequences which are cleavable bya protease associated with wound fluid such that the rate of release ofthe therapeutic agent increases in the presence of the protease.
 2. Awound dressing according to claim 1 wherein the protease is associatedwith wound infection or ulcer formation.
 3. A wound dressing accordingto claim 1 or 2 wherein the polymers themselves are not degraded by theprotease or other factors that may be present in the wound environment.4. A wound dressing according to claim 1, 2 or 3 wherein the polymer isa synthetic polymer.
 5. A wound dressing according to claim 4 whereinthe polymer is a polymer of N-(2-hydroxypropyl)methyacrylamide (HPMA).6. A wound dressing according to any one of the preceding claims whereinthe oligopeptidic sequences consist of 3 to 15 amino acids.
 7. A wounddressing according to any one of the preceding claims wherein theprotease is elastase and wherein the oligopeptidic sequence comprises orconsists of lys-gly-ala-ala-ala-lys (SEQ ID NO: 1) -Ala-Ala-Ala-,Ala-Ala-Pro-Val (SEQ ID NO: 2), Ala-Ala-Pro-Leu (SEQ ID NO: 3),Ala-Ala-Pro-Phe (SEQ ID NO: 4), Ala-Ala-Pro-Ala (SEQ ID NO: 5) orAla-Tyr-Leu-Val (SEQ ID NO: 6).
 8. A wound dressing according to any oneof claims 1 to 6 wherein the protease is a matrix metalloproteinase andwherein the oligopeptidic sequence comprises or consists of-Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO: 7), -Gly-Pro-Leu-Gly-Pro-Z- (SEQ IDNO: 8), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 9), or -Ala-Pro-Gly-Leu-Z-(SEQ ID NO: 10), where Y and Z are amino acids.
 9. A wound dressingaccording to any one of claims 1 to 6 wherein the protease is acollagenase and wherein the oligopeptidic sequence comprises or consistsof -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 11), -ProLeu-Gly-Leu-Leu-Gly-Z-(SEQ ID NO: 12), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 13),-Pro-Leu-Gly-Cys (Me)-His- (SEQ ID NO: 14), -Pro-Leu-Gly-Leu-Trp-Ala-(SEQ ID NO: 15), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 16), or-Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 17), where Z is an amino acid.10. A wound dressing according to any one of claims 1 to 6 wherein theprotease is a gelatinase and wherein the oligopeptidic sequencecomprises or consists of -Pro-LeuGly-Met-Trp-Ser-Arg- (SEQ ID NO: 18).11. A wound dressing according to any one of claims 1 to 6 wherein theprotease is thrombin and wherein the oligopeptidic sequence comprises orconsists of -Gly-Arg-Gly-Asp- (SEQ ID NO: 19), -Gly-Gly-Arg-,-Gly-Arg-Gly-Asp-Asn-Pro- (SEQ ID NO: 20), -Gly-Arg-Gly-Asp-Ser- (SEQ IDNO: 21), -Gly-Arg-Gly-Asp-Ser-Pro-Lys- (SEQ ID NO: 22), -Gly-Pro-Arg-,-Val-Pro-Arg-, or -Phe-Val-Arg-.
 12. A wound dressing according to anyone of claims 1 to 6 wherein the protease is stromelysin and wherein theoligopeptidic sequence comprises or consists of-Pro-TyrAla-Tyr-Trp-Met-Arg- (SEQ ID NO: 23).
 13. A wound dressingaccording to any one of the preceding claims wherein the therapeuticagent is an antimicrobial agent, a pain relieving agent, an antiseptic,an analgesic, a local anaesthetic, or a protease inhibitor.
 14. A wounddressing according to any one of the preceding claims wherein thetherapeutic agent is incorporated within the matrix.
 15. A wounddressing according to claim 14 wherein the wound contacting layer of thedressing may comprise or consist of the matrix within which thetherapeutic agent is incorporated.
 16. A wound dressing according toclaim 15 wherein the dressing comprises a liquid permeable woundcontacting layer, an intermediate layer comprising or consisting of thematrix within which the therapeutic agent is incorporated and an outer,liquid-impervious backing layer.
 17. A wound dressing according to anyone of claims 1 to 13 wherein the dressing comprises a barrier layerwhich comprises the matrix, the barrier layer being for initiallyseparating the therapeutic agent in the wound dressing from wound fluidwhen in use.
 18. A wound dressing according to claim 17 wherein thebarrier layer comprises an apertured sheet having a compositioncomprising the cross-linked polymers applied thereto in occlusivefashion.
 19. A wound dressing according to claim 17 or 18, wherein alayer of the therapeutic substance is provided behind the barrier layer.20. A wound dressing according to claim 19, wherein an absorbent layeris provided behind the barrier layer and the therapeutic substance isdispersed in the absorbent layer.
 21. A wound dressing according toclaim 17, wherein the barrier layer substantially encapsulates thetherapeutic substance.
 22. A wound dressing according to any one of thepreceding claims wherein the wound dressing comprises an absorbent layerand/or a backing layer.